published_or_final_version / Obstetrics and Gynaecology / Master / Master of Philosophy

Our laboratory has previously demonstrated that experimental peripheral inflammatory pain (PIP), induced by subcutaneous plantar injection of λ-carrageenan in Sprague Dawley rats, results in increased expression and activity of the ATP-dependent efflux transporter P-glycoprotein (P-gp) that is endogenously expressed at the blood-brain barrier (BBB). Increased P-gp functional expression was associated with a significant reduction in CNS uptake of morphine and, subsequently, reduced morphine analgesic efficacy. The present study examined whether the PIP-induced increase in P-gp functional expression was due to changes in intracellular trafficking (i.e., localization of P-gp), mediated by changes in the association of P-gp and caveolin-1, a key trafficking protein. These studies also determined if the drug diclofenac, a non-steroidal antiinflammatory (NSAID) that is commonly administered in conjunction with opioids during peripheral inflammatory pain (PIP), altered or modulated P-gp functional expression providing evidence of a drug-drug interaction. Confocal microscopy and subcellular fractionation revealed that under conditions of PIP, the disassembly of high-molecular weight P-gp-containing structures result in an increase in P-gp ATPase activity and changes in the localization of P-gp. Western blot analysis demonstrated further an increase in P-gp expression in rat brain microvessels following PIP induction and also after diclofenac treatment alone in the absence of PIP. Additionally, in situ brain perfusion studies showed that both PIP and diclofenac treatment alone increased P-gp efflux activity resulting in decreased radiolabeled- morphine uptake into the brain. This concurrent administration of NSAIDs and opioids in the presence of a pathophysiological stressor (i.e., pain/inflammation) may result in clinically significant drug-drug interactions that may impair the desired pharmacologic response and analgesic effects of opioids. Such interactions can lead to significant modifications to pain management in clinical settings. Therefore: The central hypothesis of this work is that the pathophysiological stressor peripheral inflammatory pain (PIP) and the pharmacological agent diclofenac modulate P-glycoprotein functional expression at the BBB. This hypothesis may be broken down further into two parts: 1) PIP induced changes in P-gp functional expression are mediated via changes in Pgp intracellular trafficking. 2) The non-steroidal anti-inflammatory drug Diclofenac, a drug commonly used to treat pain, modulates P-gp functional expression at the BBB thus decreasing morphine uptake into the CNS.

The ABC protein P-glycoprotein (Pgp, ABCB1) transports many structurally diverse substrates from the lipid bilayer. Previous studies demonstrated the importance of the membrane environment, but few have quantified these effects. In the present work, purified Pgp reconstituted into defined lipid systems was employed. Drug binding affinities were determined using Trp quenching, and drug-lipid partitioning by equilibrium dialysis. Pgp bound substrates from the bilayer with affinities in the millimolar range; both drug-Pgp and drug-lipid interactions were important. The kinetics of Pgp-mediated drug transport were sensitive to drug structure and lipid environment. The rate of transport is proposed to depend on the affinity of Pgp for substrate and conformational changes. The lipid bilayer affected the stability of Pgp catalytic activity which provided evidence for distinct basal and drug-stimulated ATPase cycles. Overall, the lipid environment had pronounced effects on Pgp-mediated drug binding, transport and catalytic functions. / Canadian Cancer Society

Anthelmintic treatment of livestock is an important aspect of the control of gastrointestinal parasites. Resistance to anthelmintics is common, and an understanding of resistance requires knowledge of an anthelmintic's mode(s) of action and mechanism(s) of resistance. The parasitic nematode, Haemonchus contortus, has developed resistance to benzimidazoles and avermectins/milbemycins. Proposed mechanisms of resistance are here supported by genetic changes observed in genes whose protein products are believed to interact with these anthelmintics. Statistically significant differences in allele frequencies were observed between untreated and ivermectin- and moxidectin-treated strains in a gene encoding a putative glutamate-gated chloride channel alpha subunit, a proposed target of avermectins/milbemycins. One allele appeared to be associated with resistance. Similar changes in allele frequencies in the same strains occurred in a gene encoding a subunit of a gamma-aminobutyric acid receptor. Significant differences in allele frequencies of a gene encoding a P-glycoprotein were found in strains of H. contortus treated with ivermectin and moxidectin compared to derived, untreated strains. In all treated strains, one allele appeared to be associated with resistance. Similarly, allele frequencies of this gene were significantly different between a cambendazole-treated strain and its derived, untreated strain. These results implicate glutamate-gated chloride channels and gamma-aminobutyric acid receptors in mechanisms of resistance to avermectins/milbemycins and implicate P-glycoprotein in a mechanism of resistance to avermectins/milbemycins and benzimidazoles in H. contortus.

Resistance to the avermectin/milbemycin class of anthelmintics in nematodes has become a serious problem worldwide due to their unrestricted usage. Resistance to these compounds is attributed to the over-expression of the transport protein, P-glycoprotein (P-gp). P-gp acts by pumping drug molecules out from the cell or organism, P-gp efflux activity can be blocked using multidrug resistance (MDR) modulators associated with chemotherapy to enhance their therapeutic effect. A series of experiments was undertaken to determine if the association of the anthelmintics, ivermectin (IVM) and moxidectin (MOX), and MDR modulators would increase the anthelmintics' efficacy against resistant parasites. Using an in vitro migration assay, IVM and MOX in the presence or absence of verapamil (VRP), CL347,099 and cyclosporin A (CyA) were used against IVM- and MOX-selected strains of H. contortus. The modulators alone had no effect on reducing the number of migrating larvae, IVM and MOX had a significant increase in efficacy of 52.7 and 58,3% respectively, when used in association with VRP, above that obtained with the anthelmintics alone. CL347,099 was also able to significantly increase the IVM and MOX efficacy by 24.2 and 38.9%, respectively. The effect of IVM and MOX in combination with VRP and CL347,099 was determined in jirds infected with selected strains of H. contortus. The combinations of VRP with either IVM or MOX significantly reduced worm counts of the selected strains compared with the untreated controls, whereas IVM or MOX alone did not. CL347,099 plus MOX combination was significantly more efficacious than moxidectin alone against the selected strains. To evaluate the effect of VRP on the pharmacokinetic behaviour of the anthelmintics IVM and MOX, the drug combination was given to sheep. The IVM plus VRP treatment resulted in an increase of the pharmacokinetic parameters of IVM. The peak concentration (83%) and area under the curve (54%) were significantly differen

Anthelmintics, antiparasitic agents, have been developed as a main weapon to control parasitic nematodes of domestic ruminants. Unfortunately, the intensive use of anthelmintics leads to the development of drug resistance in parasite populations. Anthelmintic resistance has compromised the control of nematode parasites and has become a major problem in many countries of the world. Resistance to the newest anthelmintics such as ivermectin (IVM) and related anthelmintics in Haemonchus contortus in sheep has been developing rapidly in recent years. The development of drug resistance is an evolutionary process that leads to genetic changes in parasite populations in response to drug exposure. However, the mechanism of ivermectin resistance in nematode parasites is unknown. P-glycoprotein (Pgp) has been well documented in mammalian cells as a membrane transporter by actively extruding a variety of structurally and functionally unrelated hydrophobic cytotoxic drugs out of the cell. This study was to determine whether there is an association between specific alleles at the Pgp locus and IVM or moxidectin (MOX) selection in H. contortus, by investigating the genetic variation of the Pgp homologue in unselected and IVM- and MOX-selected strains of H. contortus. (Abstract shortened by UMI.)

Myoclonus Dystonia (MD) is an autosomal dominant movement disorder characterized by bilateral myoclonic jerks paired with dystonia 1. Mutations have been mapped to the ε-sarcoglycan (SGCE) gene in about 40% of patients 2,92. The purpose of this project was to examine the properties of SGCE in the central nervous system (CNS) and use this knowledge to elucidate the pathology of MD. Although Sgce is a member of the sarcoglycan complex (SGC) in other tissues, little is known about its interactions in the CNS. The vast majority of mutations in SGCE alter the translational reading frame. Proteins arising from these rare mutations are less stable than the wild type (WT) and undergo preferential degradation via the ubiquitin proteasome system 3. As this locus is maternally imprinted, patients with MD are effectively null for sgce expression 73,91. Therefore, Sgce knock out (KO) models should approximate MD conditions both in vivo and in vitro. As there are no current treatments for MD, in sight into the pathology of the disease will aid in eventual treatments and help bring patients some relief by finally understanding their disease. Since a large percentage of MD patients are without the sgce protein, identifying what this protein’s function is and how its absence effects normal processing in the brain should help to identify the underlying cellular pathology which produces the MD phenotype. This research was performed under the hypothesis that, in neuronal cells, sgce interacts with a group of proteins that together play a role in stabilization and localization of ion channels and signaling proteins at the cell membrane. The aims were to: (1) Build a MD mouse model with either a conditional knock-out (cKO) or a conditional gene repair (cGR) mutation; (2) Use neuroblastoma cells to identify the other proteins which interact with sgce in neurons, and; (3) Determine if there is a disruption of the localization of the sgce-complex members due to the loss of sgce. Recombineering was used to complete the constructs for two transgenic mouse models: One model for the KO of exon 4 of sgce and one for the cGR in intron 1. Primary neurosphere lines from two previously generated chimeras were developed, as well as from a WT mouse. These neurosphere cell lines allowed comparisons of RT-PCR results from a heterogeneous neurological cell population to neuroblastoma cell lines. mRNA is present in neuronal cells for many of the DGC associated proteins. It was confirmed that the KD of sgce results in a reduction of nNOS protein and in increased proliferation of NIE cells. By using a nitrite/nitrate assay as well as studies with L-NAME, it was confirmed that this increased proliferation was in fact due to a lack of nNOS function. These proliferation changes did not occur in N2A cells, which do not express high levels of nNOS during proliferation, further confirming nNOS’s role in the proliferation changes. Using qRT-PCR, KD of sgce was shown to result in significant changes in the transcript levels for many DGC associated proteins. This suggests that a DGC-like complex is forming in neuronal cells. Also, as a result of difficulties with the research, it became clear that over-expression of sgce causes cell death. This observation was quantified using cell counts and TUNEL staining, both showing significant results. Additionally, several new constructs were created which will hopefully be of use for future students wanting to study sgce’s functions. New shRNA targeting sgce and sgcb have been made and both constructs result in reducing the expression of sgce. Seven different flag-tagged sgces have been created and some of these have been transferred into a tet-inducible system, which should circumvent the problem of over-expression. Finally GFP-tagged constructs for sgce and sgcb have been made and pooled clones have been developed. These tools will hopefully enable future students to continue to tease apart sgce’s function(s).

Little is known about potential influences of kinase pathway modulation on expression and activity of P-glycoprotein (P-gp). A protein kinase inhibitor (PKI) library was screened, to determine its effects on activity and expression of P-gp, in various cell lines. Cell lines were incubated with PKI for 24 h. Subsequent P-gp substrate accumulation studies were performed. Changes in P-gp activity and/or expression ≥ 25% compared to control were considered hits. Kinase pathways identified as P-gp activity hits were examined for their ability to modulate permeability. PKI families GSK-3, Craf1 and VEGFR2 and Tie-2, significantly modulated P-gp activity in the MDCK cell line. PKI families GSK-3, Iκκ and Jnk2/3 significantly modulated P-gp activity in the Caco-2 cell line. Few P-gp activity hits significantly modulated P-gp expression. PKIs modulate P-gp activity more than P-gp expression in a cell line dependent manner, excluding GSK-3 PKI family, which appears to be cell line independent.

Chemotherapy is used in the treatment of cancer. Unfortunately, drugs often fail due to multidrug resistance (MDR) caused by P-glycoprotein (P-gp1or ABCB1) and the multidrug resistance-associated protein (MRP1 or ABCC1). These proteins bind and transport drugs out of cancer cells, thereby conferring MDR. / The second chapter of this thesis addresses an unexplained phenomenon that accompanies P-gp1 expression, collaterally sensitive to verapamil. The collective results of this work demonstrated that treatment of cells that over-express P-gp1 with verapamil induces apoptosis. Furthermore, the findings show that the ATPase activity of P-gp1 was activated by verapamil. The degree of ATPase activation was proportional to the level of apoptosis and the increased demand for ATP resulted in the production of reactive oxygen species (ROS). Finally, the production of ROS led to cell death mediated by apoptosis in that experimental model system. / Chapters three and four are devoted to understanding the binding characteristics of MRP1 with two of its physiological substrates, glutathione (GSH) and leucotriene C4(LTC4). Photoreactive derivatives of these substrates were synthesised to address this objective, IAAGSH and IAALTC4. Photolabelling and transport studies showed that these derivatives have similar binding characteristics as the native compounds. In addition, photolabelling of MRP1 occurred with a high specificity with both compounds. IAAGSH and IAALTC 4 were also used to determine the locations of GSH and LTC4 binding sites. This was accomplished using MRP1-variants containing hemagglutinin (HA) epitopes at specific locations in the amino acid sequence. Through photoaffinity labelling, immunoprecipitation, and trypsin digestion, a map of binding sites for IAAGSH or IAALTC4 was obtained. Both LTC4 and GSH bound to transmembrane (TM) regions 10-11 and 16-17 which have been previously implicated in drug binding. Furthermore, novel binding sites for both substrates were discovered. IAALTC4 photolabelled a novel site within the first five TMs (TMD0) of MRP1, whereas IAAGSH labelled two cytoplasmic regions (L1 and L0). These may represent specific binding sites for LTC4 and GSH. / The work within this thesis explores some of the biochemical characteristics of Pgp1 and MRP1 that are not directly related to drug resistance and may lead to new strategies in cancer treatment.

An outline of the work contained in this thesis is presented. The first chapter is a critical review of the literature pertaining to the pathophysiological mechanisms operational with regards to the antiphospholipid syndrome (APS). The syndrome is characterised by venous and arterial thrombosis, and recurrent fetal loss, in association with the persistent presence of antibodies targeting the main autoantigen beta 2-glycoprotein I (β2GPI). The second chapter reviews the literature delineating the diverse physiological functions of β2GPI, and then relates them to its role in our current understanding of the pathophysiology of APS. The third chapter presents a critical review of the evidence base for the diagnosis and management of APS. The fourth chapter describes the interaction between β2GPI and the glycoprotein Ib alpha (GPIbα) subunit of the platelet receptor GPIb-IX-V. GPIbα is an important platelet adhesion receptor, which mediates multiple additional functions on the platelet surface, including binding coagulation factor XI (FXI). The implication of the interaction between β2GPI and GPIbα on platelet activation and the release of thromboxane in the presence of anti-β2GPI antibodies is explored, as well as the intracellular pathways via which this activation occurs. The relevance of these findings to understanding APS pathogenesis, in particular thrombosis, is discussed. The fifth chapter delineates the interaction between the fifth domain of β2GPI and FXI and its activated form factor XIa (FXIa). The ability of FXIa to cleave β2GPI between lysine (Lys) 317 and threonine (Thr) 318, and modulate its function is reported. The sixth chapter describes the ability of β2GPI to inhibit FXIa autoproteolytic hydrolysis at the specific FXIa residues arginine (Arg) 507, Arg532 and Lys539. This interaction with β2GPI stabilizes FXIa activity over time, and leads to enhanced FXIa mediated fibrin formation. This is a novel physiological function of β2GPI with important implications. Recent epidemiological studies by others have emphasized the critical role of FXIa in pathological thrombus propagation. The seventh chapter defines the relevance of the FXIa residues Arg507, Arg532 and Lys539 to FXIa mediated inactivation by the main FXIa inhibitor Protease Nexin 2 (PN2), and by Antithrombin III (ATIII). Insights into future directions for research are presented and discussed within each individual chapter.